Anti human abcg2

ABCG2⁺/ABCB5⁺ LESCs (1 × 10⁵) were seeded onto 0.4 μm 12-well transwell filters in 10% FBS DMEM. At 90% cell confluence, DMEM was replaced with CnT30 medium. Negative control cultures were maintained in 10% FBS DMEM. Cell differentiation was performed under immersed conditions. Culture media was changed every 2 days for 5 days, and then the cells were fixed in 3.7% formaldehyde and immunostained with the appropriate antibodies.
After 6 days, the transwell filters were fixed in 3.7% formaldehyde, embedded into paraffin, and sliced into 8 μm-thick sections. The sections were washed in PBS, blocked in 5% normal donkey serum and 0.1% Triton X-100, and incubated overnight in primary antibody 2 h at room temperature. The primary antibodies included anti-human p63α (Cell Signaling), anti-human ABCG2 (Abcam), anti-human ABCB5 (Invitrogen), anti-human CK3 (Abcam), and anti-human desmoglein 3 (Novus Biologicals). Then, the sections were labeled with a fluorescein-conjugated secondary antibody (Molecular Probes), and nuclei were counterstained with DAPI. Samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan).

For cell staining, cells on the coverslip were washed with PBS and fixed in 4% paraformaldehyde. For tissue staining, the whole cornea with limbus was carefully harvested and embedded in OCT compound. Eight-μm frozen sections were made and fixed in cold acetone, after which specimens were treated with 0.1% Triton X-100 for 30 min and blocked with 5% BSA for 1 h. Fluorescein conjugated or non-conjugated primary antibody at appropriate concentration was used for incubation at 4°C overnight. If necessary, specimens were then incubated with fluorescent second antibody at room temperature for 2 h. Finally, after stained with DAPI for 5 min, slides were mounted with mounting medium and observed with a fluorescence microscopy (Nikon, Tokyo, Japan). The primary antibodies used were as follows: anti-human p63, anti-human Ki67, anti-human ABCG2, anti-human CK3/12, anti-human ABCB5, anti-human SSEA4, anti-human Nanog, anti-human OCT4 (all from Abcam, Cambridge, MA), anti-rabbit CD11b, anti-rabbit CD161, anti-rabbit CD4, and anti-rabbit CD8 (all from Biolegend, San Diego, CA).